Diagnostic assays based upon the interaction of a ligand and a ligand-binding molecule are well known in the art and are widely used in clinical laboratories to detect a variety of antigens or antibodies within a biological sample. Historically, agglutination assays provided a simple way of detecting the specific interaction between an antigen and an antibody through the formation and precipitation of large immunocomplex structures.
Cells in general, and red blood cells in particular, are also amenable to a variety of agglutination methods. Conventional blood typing tests such as the direct and indirect Coomb's tests rely on the agglutination of red blood cells to determine blood group compatibility or to diagnose serious auto-immune diseases such as hemolytic anemia.
The direct Coomb's test or direct anti-globulin test, is used to detect antibodies or complement system factors bound to the surface antigens of a patient's red blood cells in vivo. In a positive Coomb's test, the addition of rabbit anti-human antibody (Coomb's reagent) to a patient's red blood cells bound by human antibody results in agglutination and precipitation of the red blood cells and is thereby indicative of autoimmune hemolytic anemia.
In the indirect Coomb's test or indirect anti-globulin test, the patient's serum is incubated with the red blood cells of a potential donor. If antibodies in the patients serum are bound to the donor's red blood cells, addition of Coomb's reagent results in agglutination and precipitation of the donor's red blood cells and thereby indicates an incapability between the patient and the potential donor's blood. Conversely, the absence of agglutination indicates that the antibodies in the patient's serum do not recognize the surface antigens on the donor's red blood cells. The donor's blood is therefore compatible and can be used for a blood transfusion.
Agglutination immunoassays while simple and cost-effective often lack the required sensitivity for the detection of minute amounts of antigen and are also prone to subjectivity in the interpretation of the results. More recently, an agglutination-type assay with increased sensitivity and reproducibility was achieved by attaching antibodies to sub-micron sized polystyrene microspheres, often called “uniform latex particles”. These Brownian particles significantly improved detection sensitivity, because of the increase in scattered light when aggregation between grafted colloids takes place. These improvements to agglutination immunoassays have led to a rapid expansion of commercially available diagnostic kits for the detection of a wide variety of disease-related antigens. Cell-based agglutination assays such as the Coomb's assay or simplified blood typing methods such as the ID-Micro Typing System, Inc., disclosed in U.S. Pat. No. 5,338,689 (Stiftung fur diagnostische Forschung), continue to provide a valuable clinical diagnostic tool especially in view of the growing demand for blood transfusions and the requirement for rapid assessment of blood types for blood transfusions in urgent care facilities.
Despite the improvements over the years, agglutination type assays, and cell-based agglutination type assays in particular, remain laborious and cumbersome because of the need for multiple washing steps and the sequential addition of reagents as dictated by the experimental protocol. Hence, these assays are not easily automated. The handling of large numbers of human patient samples, for example blood samples, also increases the risk of experimental error, reproducibility, contamination and infection of laboratory personnel with human pathogens such as HIV and hepatitis viruses B and C.
Information relevant to attempts to address these problems can be found in U.S. Pat. Nos. 4,087,248; 4,590,157; 4,775,515; 4,960,566; 4,963,498; 5,019,351; 5,144,139; 5,174,162; 5,891,740; 5,942,442; 5,976,896; 6,218,193; 6,261,847; 6,375,817; 6,517,778; the U.S. Patent Application Publication Nos. US 2003/0022382; US 2005/0048519; US 2006/0194342, the International PCT Applications Nos. PCT/US87/02054; PCT/US94/01182; PCT/GB 1999/000052; PCT/GB2005/004166; PCT/EP2005/001029; PCT/GB 1990/000202, the European patent documents EP 212314, EP 340562, EP 483117; EP 542655 and the Japanese patent documents JP 58073866, JP 62240843 and JP 2005164330. Each one of these references suffers, however, from one or more of the following disadvantages: Particle agglutination reactions requiring the repeated intervention of lab personnel, the absence of the description of a device in which all the steps required in a particle agglutination reaction can occur, the absence of automation of multiple particle agglutination reactions using such a device and the absence of a means to detect a particle agglutination reaction in such a device.
For the foregoing reasons, there is an unmet need in the art to implement the automation of particle agglutination type assays so they require minimal interaction with the operator and thereby improve the safety, reliability, cost-effectiveness and efficiency of these important clinical assays.